Effect of D240G substitution in a novel ESBL CTX-M-27

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1 Advance Access published May 29, 2003 Journal of Antimicrobial Chemotherapy DOI: /jac/dkg256 Effect of D240G substitution in a novel ESBL CTX-M-27 R. Bonnet 1 *, C. Recule 2, R. Baraduc 1, C. Chanal 1, D. Sirot 1, C. De Champs 1 and J. Sirot 1 1 Laboratoire de Bactériologie, Faculté de Médecine, Service de Bactériologie-Virologie, 28 Place Henri-Dunant, Clermont-Ferrand Cedex; 2 Laboratoire de Bactériologie, CHU de Grenoble, Chemin Maquis du Grésivaudan, La Tronche, France Received 27 January 2003; returned 14 February 2003; revised 13 March 2003; accepted 13 March 2003 Escherichia coli clinical strain Gre-1 collected in 2000 from a French hospital harboured a novel CTX-Mencoding gene, designated bla CTX-M-27. CTX-M-27 differed from CTX-M-14 only by the substitution D240G and was the third CTX-M enzyme harbouring this mutation after CTX-M-15 and CTX-M-16. The Gly-240-harbouring enzyme CTX-M-27 conferred to E. coli higher MICs of ceftazidime (MIC, 8 versus 1 mg/l) than did the Asp- 240-harbouring CTX-M-14 enzyme. Comparison of CTX-M-14 and CTX-M-27 showed that residue Gly-240 decreased K m for ceftazidime (205 versus 940 µm), but decreased hydrolytic activity against good substrates, such as cefotaxime (k cat, 113 versus 415 s 1 ), probably owing to the alteration of β3 strand positioning during the catalytic process. Keywords: CTX-M, β-lactamase, D240G mutation, ESBL Introduction The first extended-spectrum β-lactamase (ESBL) of the CTX-M type (MEN-1/CTX-M-1) was reported at the beginning of the 1990s. 1,2 Initially characterized in Europe, CTX-M-producing strains have been observed over a wide geographic area including the Near and Far East, 3 8 South America, 3,9 12 Africa 13 and Europe. 1,2,14 24 CTX-M enzymes have been observed in different species of the Enterobacteriaceae family such as: Escherichia coli, Salmonella enterica serovar Typhimurium, Klebsiella pneumoniae, Proteus mirabilis, Citrobacter freundii, Citrobacter amalonaticus, Enterobacter aerogenes and Enterobacter cloacae; and in the species Vibrio cholerae El Tor. 12 The CTX-M enzymes form a rapidly growing family that comprises currently at least 34 enzymes of which 19 have been described in the last 3 years. They are generally much more active against cefotaxime than against ceftazidime and aztreonam. 1 The flexibility of the β3 strand and Ω loop, and residues Asn-104, Ser-237, Asp-240 and Arg-276 are involved in the cefotaxime-hydrolyzing activity of CTX-M enzymes. 1,5,17 19,25,26 There have been recent reports of CTX-M mutants exhibiting an increased enzymic activity against ceftazidime: the P167S mutant of CTX-M-18 (also designated CTX-M-14), designated CTX-M-19, 21 and D240G mutants of CTX-M-3 and CTX-M-9, designated CTX- M-15 and CTX-M-16, respectively. 8,10,14 In France, we isolated a CTX-M-producing strain that produced a novel D240G variant designated CTX-M-27 and derived from CTX-M-14 as well as CTX-M-19. The biochemical characterization of the two β-lactamases CTX-M-14 and CTX-M-27 and molecular modelling give insights into the role of the D240G substitution in CTX-M enzymes. Materials and methods Clinical strain Table 1 shows the strains and plasmids used in this study. Clinical strain Gre-1 was isolated in 2000 from a patient hospitalized in Grenoble, France. E. coli transformants producing CTX-M were used for comparison. Susceptibility to β-lactams MICs were determined by a dilution method on Mueller Hinton agar (Sanofi Diagnostics Pasteur, Marnes la Coquette, France) with an inoculum of 10 4 cfu per spot. Antibiotics were provided as powders by SmithKline Beecham Pharmaceuticals, Nanterre, France (amoxicillin, ticarcillin and clavulanate), Lederle Laboratories, Paris-La Défence, France (piperacillin and tazobactam), Eli Lilly, Paris, France (cefalothin), Roussel-Uclaf, Paris-La Défence, France (cefotaxime, cefpirome), Glaxo Wellcome, Marly-le-Roi, France (ceftazidime), and Sanofi Winthrop, Gentilly, France (aztreonam). Detection of ESBLs was carried out with the standard double disc synergy tests as described previously. 27 Antibiotic discs for agar tests were obtained from Sanofi Diagnostics Pasteur.... *Corresponding author. Fax: ; Richard.Bonnet@u-clermont1.fr... Page 1 of 7 Published by Oxford University Press

2 Table 1. Strains and plasmids used in the study R. Bonnet et al. Strain or plasmid Relevant genotype and produced β-lactamase (pi) Source Strain E. coli Gre-1 clinical strain producing TEM-1 (5.4) and CTX-M-27 (8.2) this study E. coli DH5α SupE44 hsds20 (r B, m B ) reca13 ara-14 proa2 lacy1 galk2 rpsl20 xyl-5 (28) mtl-1 Plasmid pclgre-1 recombinant pbk-cmv plasmid which contains a 0.9-kb fragment encoding this study CTX-M-27 (8.2) pclcf-1 recombinant pbk-cmv plasmid, which contains a 0.9-kb fragment encoding (16) CTX-M-14 (7.9) pbk-cmv plasmid vector Stratagene Isoelectric focusing Isoelectric focusing was carried out with polyacrylamide gels containing ampholines with a ph range of as previously described. 10 β-lactamases of known pis were used as standards: TEM-1 (pi 5.4), TEM-24 (pi 6.5), SHV-1 (pi 7.6) and SHV-5 (pi 8.2). Amplification of CTX-M-encoding genes The detection of CTX-M-encoding genes was carried out with the primers CTX-MA (5 -CGCTTTG CGATGTGCAG-3 ) and CTX-MB (5 -ACCGCGATATCGTTGGT-3 ) (temperature of annealing of 54 C). They amplify a 550 bp internal fragment from positions (bla CTX-M-1 numbering), which correspond to conserved regions of bla CTX-M -type genes. The complete ORF of the bla CTX-M-27 gene was amplified with the primers CTX-M-9A (5 -CTGATGTAACACGGA- TTGAC-3 ) and CTX-M-9C (5 -AGCGCCCCATTATTGAGAG-3 ) (temperature of annealing of 54 C). β-lactamase gene cloning The recombinant DNA manipulations were carried out as described by Sambrook et al. 28 T4 DNA ligase was purchased from Boehringer Mannheim, Germany. The CTX-M-encoding sequence was cloned as follows: the complete ORF, which was amplified with proof-reading Taq polymerase Tfu (Appligene Oncor, Illkirch, France) and primers reported above, was ligated in the SmaI site of the phagemid pbk-cmv (Stratagene, La Jolla, CA, USA). E. coli DH5α 28 was transformed by electroporation. The transformants harbouring the recombinant CTX-Mencoding plasmid were selected on Mueller Hinton agar supplemented with 2 mg of cefotaxime per L. DNA sequencing The sequence was determined by direct sequencing of PCR products, carried out by the dideoxy chain termination procedure of Sanger et al. 29 on an ABI 1377 automatic sequencer using the ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with Ampli Taq DNA polymerase FS (Perkin-Elmer/Applied Biosystems Division, Foster City, CA, USA). β-lactamase preparation The CTX-M-producing E. coli DH5α was grown in 6 L of brain heart infusion broth containing cefotaxime at 2 mg/l for 18 h at 37 C. The bacteria collected by centrifugation were suspended with MES NaOH 20 mm (ph 5.5) and disrupted by ultrasonic treatment (4 30 s, each time at 20 W). After centrifugation (10 000g for 10 min at 4 C), nucleic acids were precipitated by addition of 0.2 M [7% (v/v)] spermine and centrifuged at g for 60 min at 4 C. The clarified supernatant was dialysed overnight against MES NaOH 20 mm (ph 5.5). The CTX-M purification was carried out as previously described 10 by ion-exchange chromatography with an SP Sepharose column (Amersham Biosciences Europe, Orsay, France) and gel-filtration chromatography with a Superose 12 column (Amersham Pharmacia Biotech). The total protein concentration was estimated by the Bio-Rad protein assay (Bio-Rad, Richmond, CA, USA), with bovine serum albumin (Sigma Chemical Co., St Louis, MO, USA) used as a standard. The purity of CTX-M extracts was estimated as previously described 10 by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and staining with Coomassie Blue R-250 (Sigma Chemical Co.). The renaturation of proteins and the detection of the β-lactamase activity were carried out as previously described 10 with renaturation buffer Tris HCl (100 mm) Triton X-100 [2% (v/v); ph 7.0] and 0.5 mm nitrocefin (Oxoid, Paris, France) in 100 mm phosphate buffer (ph 7.0), respectively. Determination of β-lactamase kinetic constants The steady-state kinetic parameters (K m and k cat ) of the β-lactamases were obtained by an improved computerized micro-acidimetric method derived from the technique of Labia et al. 30 using 702 SM Titrino phstat (Metrohm, Herisau, Swiss). They were determined by the analysis of the complete hydrolysis time-courses and the kinetic progress curves were fitted by non-linear least-squares regression. The concentrations of the inhibitors (clavulanate and tazobactam) required to inhibit enzyme activity by 50% (IC 50 s) were determined as previously described with penicillin G. 10 The specific activity and IC 50 s were monitored with penicillin G (200 mm) as the reporter substrate. The kinetic constants were determined three times. Sequence analysis The nucleotide sequence and the deduced protein sequence were analysed with the software available over the Internet at the National Center of Biotechnology ( A hydrophobic blot was obtained with the method of Nielsen et al. 31 Multiple sequence alignment and pairwise comparisons of sequences were carried out with the help of ClustalW 1.74 software. 32 Molecular modelling Molecular modelling was carried out using Hyperchem v6.3 software (Hypercube Inc., Gainesville, FL, USA) on the basis of the crystallographic structure of the Glu-166 Ala Toho-1 mutant 25 by introducing the mutations as part as an automated procedure. The residues, the Page 2 of 7

3 D240G substitution in CTX-M-27 Table 2. MICs for clinical strain E. coli Gre-1 and the corresponding transformant E. coli DH5α (pclgre-1) in comparison with CTX-M-14-producing transformant E. coli DH5α (pclcf-1) 16 β-lactam MICs (mg/l) for β-lactam E. coli Gre-1 (pgre-1 a ) TEM-1, CTX-M-27 E. coli DH5α (pclgre-1 b ) CTX-M-27 E. coli DH5α (pclcf-1 c ) CTX-M-14 E. coli DH5α (pbk-cmv d ) catalytic water molecule and the water molecules of the Toho-1 crystal were minimized using the Amber96 parameters 33 and a distancedependent dielectric constant by conjugate gradient energy minimization until the r.m.s. gradient was <0.1 kcal/mol. Nucleotide sequence accession number The bla CTX-M-27 nucleotide sequence data appear in the GenBank nucleotide sequence database under accession number AY Results and discussion Amoxicillin >2048 >2048 > Amoxicillin + CLA e Ticarcillin >2048 >2048 > Ticarcillin + CLA Piperacillin Piperacillin + TZB f Cefalothin > Cefuroxime <2 Cefotaxime Cefotaxime + CLA Cefpirome Cefepime Aztreonam Ceftazidime Ceftazidime + CLA a Clinical strain E. coli Gre-1 harbouring natural plasmid pgre-1, which encodes β-lactamases TEM-1 and CTX- M-27. b E. coli DH5α harbouring recombinant plasmid pclgre-1, which encodes β-lactamase CTX-M-27. c E. coli DH5α harbouring recombinant plasmid pclcf-1, which encodes β-lactamase CTX-M d E. coli DH5α harbouring plasmid vector pbk-cmv (Stratagene). e CLA, clavulanate at fixed concentration of 2 mg/l. f TZB, tazobactam at fixed concentration of 4 mg/l. Characterization of the CTX-M-27-producing clinical isolate E. coli clinical isolate Gre-1 exhibited resistance to broad-spectrum cephalosporins (MIC of cefotaxime, 256 mg/l; MIC of ceftazidime, 16 mg/l; MIC of aztreonam, 32 mg/l) (Table 2) and a positive double-disc synergy test. Isoelectric point determination with benzylpenicillin as substrate revealed the presence of two different β-lactamases (Table 1), but with cefotaxime as substrate, only one enzyme, of pi value 8.2, showed strong cefotaxime-hydrolysing activity. Cloning and DNA sequencing of β-lactamase genes The strain Gre-1 exhibited a positive amplification with primers CTX-MA and CTX-MB, which were designed to conserve sequences of bla CTX-M genes. DNA sequencing of PCR products showed that strain Gre-1 harboured a bla CTX-M-14 -type gene. The complete ORF of the bla CTX-M gene was amplified with the primers CTX-M-9A and CTX-M-9C and cloned downstream of the LacZ promoter of plasmid pbk-cmv. The sequencing of the bla CTX-M ORF revealed that strain Gre-1 harboured a new gene, designated bla CTX-M-27, which differed from bla CTX-M by the substitution A G at ORF position 725. On the basis of amino acid sequence alignment (data not shown) with CTX-M signal peptide sequences previously determined by direct amino acid sequencing 1,6 and from hydropathy plots, the deduced amino acid sequence comprised a signal peptide of 28 amino acids. Thus, the putative mature enzyme CTX-M-27 consisted of 263 amino acid residues with a calculated molecular weight of Da. This sequence differed by one or two substitutions at positions 231 and 240 (according to the numbering of Ambler et al. 34 ) from those of CTX-M-9 (A231V, D240G), CTX-M-14 (D240G) and CTX-M-16 (A231V). Thus, CTX-M-27 was the third CTX-M enzyme harbouring the substitution D240G after CTX-M-15 and CTX-M-16, 8,10, which derive from CTX-M-3 and CTX-M-9, respectively. β-lactam susceptibility of the CTX-M-27-producing E. coli transformant MICs of β-lactams for the E. coli DH5α transformants producing CTX-M-27 (pclgre-1) and CTX-M-14 (pclcf-1) 16 are listed in Table 2. The CTX-M-producing strains exhibited a high level of resistance to amino- and carboxy-penicillins (MICs > 2048 mg/l), piperacillin (MIC, 256 mg/l), cefalothin and cefuroxime (MICs, mg/l). Similar levels of resistance to cefotaxime (MICs, 16 mg/l) and aztreonam (MICs, 4 8 mg/l) were observed. However, the Gly-240-harbouring enzyme CTX-M-27, like CTX-M-15 Page 3 of 7

4 R. Bonnet et al. Table 3. Substrate profile of β-lactamases CTX-M-14, 16 and CTX-M-27 CTX-M-27 CTX-M-14 Substrate k cat (s 1 ) K m (µm) k cat /K m (µm 1 s 1 ) k cat (s 1 ) K m (µm) k cat /K m (µm 1 s 1 ) Penicillin G 11 ± ± ± ± Amoxicillin 5 ± ± ± ± Ticarcillin 1 ± ± ± ± Piperacillin 9 ± ± ± ± Cefalothin 232 ± ± ± ± Cefuroxime 79 ± ± ± ± Cefotaxime 113 ± ± ± ± Cefpirome 63 ± ± ± ± Ceftazidime 3 ± ± ± ± Aztreonam 0.4 ± ± ± ± Figure 1. Electrophoresis analysis of CTX-M-27 purified extracts. (a) SDS PAGE stained with Coomassie Brilliant Blue R-250. (b) Zymogram detection of β-lactamase activity with nitrocefin after renaturation treatment of SDS PAGE. Lanes: 1, protein molecular mass reference; 2, purified extract of β-lactamase CTX-M-27; 3, clarified extract of β-lactamase CTX-M-27. and CTX-M-16, 8,10 exhibited higher MICs of ceftazidime than its parental enzyme CTX-M-14 (8 versus 1 mg/l), which contains the residue Asp-240. Clavulanate and tazobactam restored partially or totally the activities of the β-lactams (MICs, mg/l). Kinetic constants The purified protein appeared on SDS polyacrylamide gels as a band of 28 kda for CTX-M-27 (Figure 1). The specific activity of purified ( 97% pure) CTX-M-27 was 22 µmol min 1 mg 1 of protein with 200 mm benzylpenicillin as the substrate. The kinetic constants of CTX-M-14 and CTX-M-27 are shown in Table 3. Common enzymic features were observed. There were better affinities for penicillins (K m, 6 48 µm) than for cephalosporins, cefalothin was the best substrate (k cat for cefalothin 10- to 20-fold higher than that for penicillin), and k cat for cefotaxime ( s 1 ) was higher than for ceftazidime (3 s 1 ) and aztreonam ( s 1 ). The hydrolytic properties of enzyme CTX-M-27 were therefore similar to those of previously reported CTX-M enzymes with regard to the higher catalytic activity against cefotaxime than against ceftazidime and aztreonam. However, CTX-M-27 had lower k cat values than CTX-M-14 with all substrates except ceftazidime for which the hydrolytic activity was closely related to that observed with previously reported CTX- M. 1,6,7,10,15,16,18 Shimamura et al. 26 reported an interaction between the Asp-240 side chain and the amino-thiazole ring of cefotaxime in the crystallographic acyl-intermediate of Toho-1, which accounts for its activity against cefotaxime. Residue Gly-240, which is devoid of side chains, is unable to establish this interaction and may therefore decrease the hydrolytic activity of CTX-M-27 against cefotaxime and against substrates devoid of the amino-thiazole ring such as penicillins, cefuroxime and cefalothin. The Gly-240-harbouring enzyme CTX-M-27, as previously observed with CTX-M-16, 10 had a lower K m value than the Asp-240- harbouring enzyme CTX-M-14, with regard to ceftazidime. This decrease in K m could explain the higher MIC value of ceftazidime observed for the CTX-M-27-producing E. coli than for the CTX-M- 14 producer. In TEM and SHV ESBLs, residues Lys and Arg at position 240 are known to increase the enzymic activity against ceftazidime. Lys and Arg are positively-charged residues that can form an electrostatic bond with the carboxylic acid group on oxyimino substituents of ceftazidime. 35,36 In CTX-M-27, neutral residue Gly-240 is not able to form electrostatic interactions with β-lactams but could favour the accommodation of ceftazidime. In Asp-240-harbouring enzyme CTX-M-14, the acyl-amide group of ceftazidime is probably sterically encumbered by the side chain of Asp-240, which, as a result, could alter the interactions between the ceftazidime C-7β-amid group and residues Ser-237 and Asn-132. The residue Gly-240, which is devoid of side chains, could decrease this steric hindrance. Substitution D240G did not modify the susceptibility to β-lactamase inhibitors. The two enzymes CTX-M-14 and CTX-M-27 were susceptible to tazobactam (IC 50, and µm, respectively), clavulanate (IC 50, and µm, respectively) and, to a lesser extent, sulbactam (IC 50, 3.5 and 3.4 µm, respectively). Page 4 of 7

5 D240G substitution in CTX-M-27 Figure 2. Structure of enzymes CTX-M-14 (a), and CTX-M-27 (b) obtained by molecular modelling and the crystallographic structure of the TEM-1 enzyme (c). 35 Two black dots indicate the positions of the water molecules: W1, the catalytic water molecule maintained by the lateral chains of residues 166, 170 and 70; and W2, the water molecule of the oxyanionic hole formed by the main chains of residues 70 and 237. The hydrogen bonds between residues 170, 240 and 270 are indicated by dashed lines. Molecular modelling Ibuka et al. reported that the β3 strand of CTX-M enzymes has numerous Gly residues and is therefore probably more flexible than that of TEM penicillinases. 25 Residue Gly-240 in CTX-M-27 may further increase the flexibility of the β3 strand and alter its positioning during the catalytic process. To investigate the diminution of the hydrolytic activity associated with residue Gly-240 in CTX-M-27, the enzymes CTX-M-14, CTX-M-27 and CTX-M-1 were modelled by geometric optimization after the introduction of amino acid substitutions and of the catalytic water molecule in the crystallographic structure of Toho-1. The major differences observed between the models obtained and the TEM-1 crystallographic structure (Figure 2c) 37 are shown in Figure 2 (a and b). In the CTX-M enzymes, the loop connecting the β5 strand and the H11 helix exhibited two additional residues and was oriented towards the C-terminal extremity of the β3 strand unlike that in the TEM-type enzyme. In the CTX-M-14 model, the residue Asn-270 of this loop established a hydrogen bond with the Asp-240 side chain (Figure 2a). This interaction could favour the correct positioning of the β3 strand residues during the catalytic process. In the crystal structure of Toho-1 acyl-enzyme intermediates, 26 the interaction between residues 270 and 240 is mediated by a water molecule. However, the calculated energy from the structure exhibiting indirect binding was higher than that obtained from the structure exhibiting direct interaction of residues 270 and 240. No interaction between residues 240 and 270 was seen in CTX-M enzymes belonging to the CTX-M-1 group, because they are devoid of residue Asn-270. However, these enzymes harbour residue Lys-271, which could interact with residue Asp-240, and could replace the Asn-270 (data not shown). Following the substitution D240G, interaction between the residues 240 and 270 or 271 was absent in CTX-M-27 (Figure 2b). The absence of this interaction in CTX-M-27 could work towards modifying the flexibility of the C-terminal of the β3 strand. During the catalytic process, this flexibility could favour the accommodation of substrates, but could alter the geometry of the oxyanionic hole and/ or the positioning of the catalytic water molecule as the result of modifications of the interactions between residues 240 and Asn-170. Page 5 of 7

6 R. Bonnet et al. In conclusion, residue Gly-240 in CTX-M-27 decreases the K m for ceftazidime but could decrease hydrolytic activity against good substrates, probably by modifying β3 strand-residue positioning during the hydrolytic process. Acknowledgements We thank Rolande Perroux, Marlène Jan and Dominique Rubio for technical assistance. This work was supported in part by a grant from the Ministère de l Education Nationale, de la Recherche et de la Technologie. References 1. Barthélémy, M., Péduzzi, J., Bernard, H., Tancrede, C. & Labia, R. (1992). Close amino acid sequence relationship between the new plasmidmediated extended-spectrum β-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca. Biochimica Biophysica Acta 1122, Bauernfeind, A., Casellas, J. M., Goldberg, M., Holley, M., Jungwirth, R., Mangold, P. et al. (1992). A new plasmidic cefotaximase from patients infected with Salmonella typhimurium. Infection 20, Bauernfeind, A., Stemplinger, I., Jungwirth, R., Ernst, S. & Casellas, J. M. (1996). Sequences of β-lactamase genes encoding CTX-M-1 (MEN-1) and CTX-M-2 and relationship of their amino acid sequences with those of other β-lactamases. Antimicrobial Agents and Chemotherapy 40, Chanawong, A., M Zali, F. H., Heritage, J., Xiong, J. H. & Hawkey, P. M. (2002). Three cefotaximases, CTX-M-9, CTX-M-13, and CTX- M-14, among Enterobacteriaceae in the People s Republic of China. Antimicrobial Agents and Chemotherapy 46, Ishii, Y., Ohno, A., Taguchi, H., Imajo, S., Ishiguro, M. & Matsuzawa, H. (1995). Cloning and sequence of the gene encoding a cefotaxime-hydrolyzing class A β-lactamase isolated from Escherichia coli. Antimicrobial Agents and Chemotherapy 39, Ma, L., Ishii, Y., Ishiguro, M., Matsuzawa, H. & Yamaguchi, K. (1998). Cloning and sequencing of the gene encoding Toho-2, a class A β-lactamase preferentially inhibited by tazobactam. 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